mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.

. Analysis of HIS-72, EGL-1, and PTL-1 split wrmScarlet tags in C. elegans: (A) Plasmid maps for the mScarlet knock-in constructs developed as part of this project. pGLOW39 and pGLOW63 allow tagging of any gene of interest (GOI) with mScarlet-I-C1 or split fluorophore wrmScarlet 11 , respectively. ccdB regions are removed by DNA digest and homology arms for the GOI are cloned as described by Dickinson et al. 2015. pGLOW119 allows cloning of any promoter upstream of split fluorophore wrmScarlet [1][2][3][4][5][6][7][8][9][10] , and this construct can be inserted via CRISPR/Cas9 homologous recombination into the ttTi5605 locus on LGII. (B) Testing the split fluorophore system using HIS-72. (Top) Endogenous expression of tiny-tagged HIS-72 (his-72::3xMyc::wrmScarlet 11 in an muIs252 (Peft-3::wrmScarlet 1-10 ) genetic background. HIS-72 is visible in all somatic nuclei, consistent with Goudeau et al. 2021. This confirms that the 3xMyc epitope tag does not interfere with complementation of the split fluorophore. (Middle) We created an alternative somatic expression construct for wrmScarlet 1-10 using the his-72 promoter and tbb-2 (3'UTR) rather than the eft-3 promoter and unc-54 (3'UTR). Both somatic wrmScarlet 1-10 constructs appear to have ubiquitous expression in all nuclei (compare top and middle panels). (Bottom) We also created a construct for neuronal expression of wrmScarlet 1-10 using the rab-3 promoter. Note that HIS-72::wrmScarlet 11 and wrmScarlet 1-10 complementation is visible in the neuronal nuclei of the head and ventral nerve cord. (C) egl-1 functions within the V5.paa lineages to eliminate the sister cell of PVD via programmed cell death. (D) The EGL-1 tiny tag is functional. We confirmed that the PVD sister cells undergo apoptosis in animals expressing egl-1::wrmScarlet 11 and Peft-3::wrmScarlet 1-10 . The dopaminergic neuron marker Pdat-1::gfp was used to identify PDE neurons and "undead" PVD sister cells in L3 worms. The relevant cell bodies are circled in white. (E) Animals with wild type EGL-1 or tiny-tagged EGL-1 have just 2 PDE neurons, whereas egl-1 knockout worms have an average of 3 (but sometimes 4) PDE and PDE-like neurons (p<0.0001, unpaired t-test). (F) To observe EGL-1 tiny tag expression, we induced programmed cell death in germ cells using UV irradiation. At 6 hours post-UV, we were unable to observe any expression in the germline (mitotic region nor death zone). Yellow arrowheads indicate out-of-focus, round, autofluorescent gut granules (i.e. halo effect). (G) N-terminal tiny-tagging strategy for the ptl-1 gene. Isoforms A-C share a common first exon, so we opted to do a 5' knock-in of wrmScarlet 11 at this exon to simultaneously tag multiple isoforms of PTL-1. (H) Mechanosensation indicates the PTL-1 tiny tag is functional. Animals bearing tiny-tagged PTL-1(N) responded to gentle touch just as well as animals bearing wild-type PTL-1 (p>0.05, paired t-tests). (I) PTL-1 is expressed in the axons of posterior mechanosensory neurons PVM, PLMR, and PLML. (Top) DIC image of the tail of a young adult worm, with the structures of PVM, PLMR, and PLML superimposed diagrammatically in black. Circles represent the cell bodies, whereas lines represent the axons. (Bottom) We were unable to observe any PTL-1(N) expression in the posterior mechanosensory neurons of wrmScarlet 11 ::ptl-1(N); Prab-3::wrmScarlet 1-10 animals using epifluorescence microscopy at 40x, the highest resolution available in our laboratory. Description mScarlet is currently our first-choice red fluorescent protein for visualizing gene expression in vivo (Bindels et al. 2017). Compared to other RFPs like mCherry or mKate2, mScarlet is less prone to unwanted oligomerization and yields superior brightness and photostability (Bindels et al. 2017). mScarlet was subsequently optimized for C. elegans expression by two independent research groups as wrmScarlet (El Mouridi et al. 2017) or mScarlet-I-C1 (Dickinson et al. 2017;Dickinson et al. 2018). Although wrmScarlet and mScarlet-I-C1 are both available via Addgene, the only available version of the former lacks synthetic introns to improve expression and the latter is available only as part of the SapTrap system for CRISPR/Cas9 knockin (pZCS16 and pMS050, respectively; Schwartz and Jorgensen 2016;Dickinson et al. 2018;Stevenson et al. 2020). Thus, to expand the mScarlet toolkit in C. elegans, we cloned mScarlet-I-C1 into the self-excising cassette (SEC) ccdB-based vector system for CRISPR/Cas9 knock-in (Dickinson et al. 2015), and this plasmid is now available via Addgene as pGLOW39 (Fig.  1A). We have tagged several proteins with mScarlet-I-C1 with excellent results and some of these strains are already available via the CGC.
Despite the improved performance of contemporary monomeric fluorophores like mScarlet and mNeonGreen (Heppert et al. 2016;Bindels et al. 2017), sometimes the relatively large size of these tags can disrupt the function of the targeted protein. In this situation, a split fluorophore approach can be utilized, where the functional fluorescent protein tag is asymmetrically split into a small polypeptide that serves as the tag (i.e. "tiny tag") and a large protein fragment that is conditionally disabled by the split (Cabantous et al. 2005). The two components must then be co-expressed to complement and yield fluorescence. wrmScarlet was recently redeveloped as a split fluorophore referred to as wrmScarlet 1-10 (large fragment) and wrmScarlet 11 (tiny polypeptide tag) (Goudeau et al. 2021). We cloned wrmScarlet 11 into the SEC system for versatile N-or C-terminal tagging of any protein of interest, and this plasmid is now available via Addgene as pGLOW63 (Fig. 1A). As with other SEC plasmids (Dickinson et al. 2015), an in-frame epitope tag is included (in this case 3xMyc) to allow purification or immunostaining of the tiny-tagged target protein. We confirmed that the epitope tag did not disrupt the interaction of the split fluorophore by examining the endogenous expression of tiny-tagged HIS-72 in an muIs252 (Peft-3::wrmScarlet 1-10 ) genetic background (Goudeau et al. 2021). Indeed, the complemented RFP was visible in all somatic nuclei via epifluorescence (Fig.  1B, top).
To investigate the potential of tiny tagging, we opted to tag three widely-studied proteins that are either small -EGL-1 (106 aa) and HIS-72 (151 aa) -and/or not fully functional when tagged with conventional full-length fluorophores (EGL-1 and PTL-1) (Krieg et al. 2017). We find that these tiny-tagged proteins appear to be fully functional, but in most cases we were unable to observe the expression of the endogenous tiny tag in vivo via epifluorescence. We first investigated the pro-apoptotic protein EGL-1. To confirm that tiny-tagged EGL-1 is functional, we examined the number of dopaminergic PDE neurons in L3 larval worms using a Pdat-1::gfp reporter (Davies et al. 2003). In egl-1 null mutants, worms have additional PDE-like neurons as a result of blocked developmental cell death in the V5.paa neuronal lineage (Fig. 1C). Tiny-tagged EGL-1 animals have a wild-type number of PDE neurons, consistent with tiny-tagged EGL-1 being a fully functional protein (Fig. 1D,E). To visualize tiny-tagged EGL-1 expression, we exposed young adult worms to UV irradiation and screened for expression of the tag in pachytene germ cells undergoing DNA damage-induced apoptosis in the so-called "death zone" (Stergiou et al. 2007;Gartner et al. 2008). Unfortunately, we were unable to observe tiny-tagged EGL-1 expression anywhere in the germline of UV-treated worms at the timepoint egl-1 mRNA levels are known to peak (6-12 hours post-UV, Stergiou et al. 2007) (Fig. 1F). Although use of transgenic egl-1 reporter genes has been successful (e.g. Johnsen and Horvitz 2016), we suspect that endogenous egl-1 expression is too transient or limited to observe in vivo. We next investigated PTL-1, the ortholog of the human tau/MAP2 protein implicated in Alzheimer's disease. ptl-1 has a somewhat complex gene structure encoding four isoforms of PTL-1, so we opted for an N-terminal tiny tag that tags three of the four isoforms (Fig. 1G). We call this set of proteins wrmScarlet 11 ::PTL-1(N). To test the functionality of tiny-tagged PTL-1(N), we examined sensitivity to gentle touch because loss of proper PTL-1 function has previously been reported to impair gentle touch sensation (Gordon et al. 2008). The six neurons which mediate this sensation are particularly enriched with ptl-1, making them attractive targets to investigate. As with EGL-1, tiny-tagging did not appear to disrupt the function of PTL-1 (Fig. 1H), but we were unable to observe tiny-tagged PTL-1(N) expression in any mechanosensory neurons (Fig. 1I). This result was unexpected, but perhaps N-terminal tiny tags do not complement as well a C-terminal tiny tags or the fraction of PTL-1(N) complemented with wrmScarlet 1-10 is not enriched enough at microtubules to observe. Overall, our findings suggest that tiny tags are generally innocuous enough to not disrupt the function of low MW endogenous proteins, but that the split fluorophore system is often not sensitive enough to allow observable expression of the complemented proteins via endogenous tags.
Overall, an important goal of the Glow Worms undergraduate research stream is to create and share resources with the C. elegans research community. Here, we have created several new plasmids and worm strains useful for CRISPR/Cas9 fluorescent protein knock-in. We anticipate that by expanding the CRISPR reagent toolkit, Glow Worms can have a meaningful and lasting impact on basic biological research, as well as foster a commitment to open and collaborative science in our students at a very early stage of their academic careers.

Plasmid construction
pGLOW39 was constructed via Gibson assembly of PCR fragments amplified from parent vectors pDD287 (SEC^ccdB^AmpR^ccdB backbone; Addgene #70685) and pMS050 (mScarlet-I-C1; Addgene #91826). pGLOW63 was constructed via Gibson assembly of PCR fragments from parent vector pGLOW39 and the wrmScarlet 11 tiny tag sequence was cloned into the plasmid via PCR primer. pGLOW119 was constructed via Gibson assembly of PCR fragments amplified from parent vector pAP087 (ttTi5605^SEC^ccdB^mKate2 backbone) and worm gDNA from strain CF4582 (wrmScarlet 1-10 ). All primer sequences available by request.

Cloning a promoter into pGLOW119
Digest (removes ccdB) SpeI Gibson assembly sequence fwd primer ATGATGGTAGCAAACTCACTTCGTccggca Gibson assembly sequence rev primer CTTGATGACTGCTTCTCCCTTCGATACCAT

Confocal imaging of the egl-1 split fluorophore following UV irradiation
Synchronized young adult worms of strain GLW71 glh-1(sam140[glh-1::T2A::wrmScarlet 1-10 ]);  ]) were irradiated with 100 J/m 2 UV-C using a Stratalinker 1800 as previously described (Stergiou et al. 2007). The presence of dead embryos on the plate at 24 hours post-UV was used as a positive control that UV treatment was yielding DNA damage. At 6-12 hours post-UV, adult worms were mounted for iSim confocal microscopy using a 60x oil objective and Kinetix camera. We were unable to observe split wrmScarlet complementation and fluorescence using this system.

Analysis of mechanosensation in ptl-1 genetic backgrounds
Assays for gentle touch sensitivity were done as previously described (Chalfie et al. 2014). Briefly, each worm was touched (i.e. tapped) a total of 5 times on the head and 5 times on the tail to trigger posterior or anterior movement, respectively. A total of 20 worms were assayed for each genotype. If the worm moved following a tap, the worm was sensitive to that tap. Data represents the average number of times (out of 5) the worm responded to a tap.